Normally, the Wnt/beta-catenin pathway controls developmental processes and homeostasis, but abnormal activation of this pathway is a frequent event during the development of cancer. The key mechanism in regulation of the Wnt/beta-catenin pathway is the amino-terminal phosphorylation of beta-catenin, marking it for proteasomal degradation. Here we present small-molecule-based identification of protein kinase C (PKC)-mediated beta-catenin phosphorylation as a novel mechanism regulating the Wnt/beta-catenin pathway. We used a cell-based chemical screen to identify A23187, which inhibits the Wnt/beta-catenin pathway. PKC was activated by A23187 treatment and subsequently phosphorylated N-terminal serine (Ser) residues of beta-catenin, which promoted beta-catenin degradation. Moreover, the depletion of PKCalpha inhibited the phosphorylation and degradation of beta-catenin. Therefore, our findings suggest that the PKC pathway negatively regulates the beta-catenin level outside of the Wnt/beta-catenin pathway.